African Swine Fever Virus MGF110-7L Induces Host Cell Translation Suppression and Stress Granule Formation by Activating the PERK/PKR-eIF2α Pathway

ABSTRACT African swine fever (ASF) is a highly contagious and often lethal disease of pigs caused by ASF virus (ASFV) and recognized as the biggest killer in global swine industry. Despite exhibiting incredible self-sufficiency, ASFV remains unconditionally dependent on the host translation machinery for its mRNA translation. However, less is yet known regarding how ASFV-encoded proteins regulate host translation machinery in infected cells. Here, we examined how ASFV interacts with the eukaryotic initiation factor 2α (eIF2α) signaling axis, which directs host translation control and adaptation to cellular stress. We found that ASFV MGF110-7L, a previously uncharacterized member of the multigene family 110, remarkably enhanced the phosphorylation level of eIF2α. In porcine alveolar macrophage 3D4/21 and porcine kidney-15 cells, MGF110-7L triggered eIF2α signaling and the integrated stress response, resulting in the suppression of host translation and the formation of stress granules (SGs). Mechanistically, MGF110-7L-induced phosphorylation of eIF2α was mediated via protein kinase R (PKR) and PKR-like endoplasmic reticulum (ER) kinase (PERK), and this process was essential for host translation repression and SG formation. Notably, our subsequent analyses confirmed that MGF110-7L was overwhelmingly retained in the ER and caused a specific reorganization of the secretory pathway. Further proteomic analyses and biochemical experiments revealed that MGF110-7L could trigger ER stress and activate the unfolded protein response, thus contributing to eIF2α phosphorylation and translation reprogramming. Overall, our study both identifies a novel mechanism by which ASFV MGF110-7L subverts the host protein synthesis machinery and provides further insights into the translation regulation that occurs during ASFV infection. IMPORTANCE African swine fever (ASF) has become a socioeconomic burden and a threat to food security and biodiversity, but no commercial vaccines or antivirals are available currently. Understanding the viral strategies to subvert the host translation machinery during ASF virus (ASFV) infection could potentially lead to new vaccines and antiviral therapies. In this study, we dissected how ASFV MGF110-7L interacts with the eIF2α signaling axis controlling translational reprogramming, and we addressed the role of MGF110-7L in induction of cellular stress responses, eIF2α phosphorylation, translation suppression, and stress granule formation. These results define several molecular interfaces by which ASFV MGF110-7L subverts host cell translation, which may guide research on antiviral strategies and dissection of ASFV pathogenesis.

However, I have a few queries as follows 1. In Fig. 1A, please provide the statistical data on RLuC activity in presence of all the tested plasmids, as shown in the presence of MGF110-7L. 2. Fig. 1A legend, please mention the concentration of different vectors expressing different members of the MGF110 family used in the experiment. 3. It is unclear how the % ATF4-EGFP expression is measured or calculated in Fig. 1B. Is this % of cells expressing EGFP or the total % of EGFP expression? Please elaborate on how the analysis is done and the percentage is calculated in the figure legend or material and methods section. 4. In Fig.1D, WB shows relatively higher expression of ATF4 in vector alone than 0.5/1 ug MGF110-7L transfected cells whereas the beta-actin seems similar, please explain or provide a low exposure image of beta-actin. 5. In Fig. 3 B and C, the effect of eif2α phosphorylation in SG formation upon arsenite treatment was reversed using DP71L whereas MGF110-7L induced SG formation was reversed using ISRIB. Ideally, the same inhibitor should be used in positive and test treatment. Please explain. 6. Please provide the intensity band ratios for Fig. E and F. 7. In line 274, the authors have mentioned that MGF-110 7L barely colocalizes with peroxi-marker, which is confusing to interpret. The fluorescence intensity graph seems to suggest partial localization, similar to Golgi marker. Please explain and also clear in the text. 8. In line 264, the heading suggests the involvement of ASFV MGF 110-7L in the secretory pathway, whereas no direct experiments have been done to prove the same. Localization of MGF 110-7L with different secretory compartments doesn't clearly suggest its involvement/role in the pathway. The heading (Line 264) should be modified. However, the possible reasons and functions related to their cellular distribution can be extrapolated in the results/discussion. 9. Authors have not mentioned anywhere the no. of biological replicates or how many times the Co-IP experiments have been repeated to conclude the MGF 110-7L host interactome shown in Fig. 7. It seems the data is obtained from only one biological experiment/ sample. Please provide detailed information about how the final list of interacting proteins is obtained for GO analysis. 10. Line 287, suggests the involvement of ASFV MGF110-7L in the modulation of ER redox homeostasis, etc., whereas authors have identified the MGF110-7L host interactors which are involved in ER redox homeostasis. The establishment of the direct involvement of MGF110-7L in ER-redox homeostasis needs further detailed experiments. Please modify the heading. 11. Please provide the species-specific information in table 1. 12. Please provide the concentration of PDIA3-HA, TMED4-HA, or PSMA4-HA plasmid in Fig. 8 legend. Please also make sure that the concentration of all the plasmids used in the study is provided in the manuscript.
Reviewer #3 (Comments for the Author): The manuscript submitted by Shichong Han et al. entitled "African swine fever virus MGF110-7L induces host cell translation suppression and stress granule formation by activating the PERK/PKR-eIF2α pathway" aims to evaluate and characterize the role of an ASFV viral protein in the infection cycle. In the Introduction section the authors wrote that "there are still no protective vaccines" which is not true. They should correct this point and add a very recent reference about ASFV vaccines (10.1080/22221751.2022.2108342). Additionally, the authors should briefly introduce some knowledge already known about the ASFV RNA helicases (e.g. 10.1080/22221751.2019.1578624 ).
In the results's section, authors should present the major limitions of using HEK293T cells to quantify the regulation of the regulation of translation rate of ATF4-RLuc, since the ASFV replication primarly occurs in macrophages.
In fig 1. please identify the different graphs, some are missing.

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11.
Please provide the species-specific information in table 1.

12.
Please provide the concentration of PDIA3-HA, TMED4-HA, or PSMA4-HA plasmid in Fig. 8 legend. Please also make sure that the concentration of all the plasmids used in the study is provided in the manuscript.

Responses to Reviewers' Comments on Manuscript ID Spectrum03282-22
We sincerely thank anonymous reviewers for their vigorous and thoughtful comments of the manuscript. We have worked seriously with the comments and believe that this version of the manuscript is greatly improved.

Reviewer comments:
Reviewer #1 (Comments for the Author): The The study is well designed and the manuscript is also very well written.
Specific Comments: 1. In Fig  In addition, we replaced the previous Fig. 1D with a version of better quality (see the new 6. Please provide the intensity band ratios for Fig. 4E  In addition, we have modified the Fig. 4E and F and the corresponding legend in the revised manuscript (see below).  Thanks again for your careful review and for pointing out these mistakes. Also, we have modified the Fig. 6

TABLE 1 RT-qPCR primers used in this study
12. Please provide the concentration of PDIA3-HA, TMED4-HA, or PSMA4-HA plasmid in Fig.   8 legend. Please also make sure that the concentration of all the plasmids used in the study is provided in the manuscript.

Response: We greatly appreciate the critical comments. Based on your suggestions, we
added the concentration of PDIA3-HA, TMED4-HA, or PSMA4-HA plasmid in the revised Fig.   8 legend (see page 40 lines 951-962). In addition, we carefully checked the whole manuscript, and we ensured that the concentrations of all the plasmids used in the study were shown in the revised Figure Legends section (see page 36-41 lines 837-983).

Reviewer #3 (Comments for the Author):
The    3. In fig 1. please identify the different graphs, some are missing.
Response: We greatly appreciate your valuable comment. Based on your suggestion, we carefully checked the previous Fig. 1 and modified it to include all the essential information.
Please see the revised Fig. 1 below.
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